Anti-tyrosinase and Anti-butyrylcholinesterase Quinolines-Based Coumarin Derivatives: Synthesis and Insights from Molecular Docking Studies

In this work, a series of anti-tyrosinase and anti-butyrylcholinesterase coumarin derivatives 4a–f and 5a–f were synthesized starting from 4-hydroxycoumarin. The condensation of 2-(arylimin)-4-hydroxycoumarins 3a–f with dimethylformamide dimethyl acetal (DMF-DMA), used as a key reaction, afforded the precursors 4a–f, whose acid treatment led to the formation of 5a–f. These prepared heterocycles were characterized by spectroscopic means including 1H-NMR, 13C-NMR, and DCI-HRMS. Their anti-tyrosinase and anti-butyrylcholinesterase activities have been evaluated in vitro and some of them exhibited promising activity supported by the molecular docking analysis to estimate possible interactions between these compounds and active sites of both proteins tyrosinase (PDB: 2Y9W) and butyrylcholinesterase (PDB: 4TPK).


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss, behavioral abnormalities, and cognitive impairments [1]. Generally, individuals with this type of neurological disorder are elderly. The ultimate cause of memory deterioration is acetylcholine and butyrylcholine deficiency [2] in the parts of the central nervous system that mediate learning and memory functions. Many therapeutic approaches have been taken in an attempt to discover agents to treat and prevent AD [3]. Despite the development of therapies, treatment is still unsatisfactory, because of the limited efficiency of BChE inhibitors, including Tacrine [4,5], Rivastigmine [6], and Galanthamine [7]. Nitrogen heterocyclic compounds, such as quinoline derivatives, can act as effective anticholinesterase agents [8], and may substantially improve AD symptoms [9][10][11]. Recently, many reports covering the development in quinoline synthesis have been published [12][13][14][15]. Quinolines, such as chloroquinine and quinine salicylate were also identified as potent inhibitors of tyrosinase [16]. The latter is a key enzyme involved in melanin biosynthesis, which is responsible for skin pigmentation [17] and plays an important role in protection against UV radiation. Many studies have shown that pigmentation disorders due to high levels of melanin can cause serious dermatological damages [18]. Skin damage is a major reason behind restricted usage of certain pharmaceuticals.
Previous research revealed that 3-substituted coumarin derivatives are very attractive scaffolds for the development of therapeutic compounds [19][20][21][22]. In addition, their anticholinesterase and anti-Alzheimer abilities are wellknown [23]. A review of structure-activity relationships (SARs) indicated that chromone compounds possess good tyrosinase inhibitory potential due to their structural similarity to flavonoids, which combat pigment disorders [24].
Building on previous research, we decided to merge two pharmacophores: a differently substituted quinoline moiety to a coumarin entity, with the aim to discover original hybrid bioactive agents that inhibit tyrosinase and butyrylcholinesterase enzymes, and to discuss the structure-activity relationship (SAR) using molecular docking analysis. With this in mind, a series of substituted 4-hydroxy-3-(quinolin-2-yl)-2H-chromen-2-one derivatives 5a-f were synthesized.

3 2 Experimental Section
DCI-HRMS (Desorption Chemical Ionization-High Resolution Mass Spectrometry) has been run in a GCT Premier Mass Spectrometer (Waters). 1 H (300 MHz) and 13 C (75 MHz) NMR spectra have been recorded on a Bruker AM-300 spectrometer, using CDCl 3 or DMSO-d 6 as solvent and none deuterated residual solvent as internal standard. Chemicals shifts (δ) are given in parts per million (ppm) and coupling constants (J) in Hertz. Melting points have been determined on a Büchi 510 apparatus using capillary tubes and are uncorrected.

General Procedure for the Synthesis of Compound 2
4-hydroxy-2H-chromen2-one (1, 4 g, 24.8 mmol) was dissolved in anhydride acetic (15 mL) and pyridine (5 mL) at room temperature. The resulted solution was brought to refux temperature for 45 min. After cooling, the reaction crude was precipitated and collected by filtration after several washing with distilled water. The recuperated yellow solid was then recrystallized from ethanol yielding compound 2 as white crystals.

General Procedure for the Synthesis of Compounds 4a-f and 5a-f
Five hundred milligrams (0.002 mol) of 3-acetyl-4-hydroxycoumarin 2 and 227 mg (0.002 mol) of mono-substituted anilines were added to a 100 mL flask in 50 mL of ethanol. After 2 h of reflux, imines 3 were formed; they were filtered and washed with ethanol then treated with 2 eq. of dimethylformamide dimethyl acetal (DMF-DMA) (0.3 mL) in toluene (5 mL) for 10 min to form the enaminic intermediate 4 [25,26]. The reaction was visualized using thin-layer chromatography (Elution System: EtOAc) showing the disappearance of the starting material and the appearance the major polar product. After evaporation of toluene, intermediates 4a-f (obtained by precipitation), were brought to reflux in 5 mL of acetic acid for 1 h. The reaction crude was then precipitated in distilled water and collected by filtration to afford compounds 5a-f (See supplementary materials for spectral data of compounds 4a-f and 5a-f).

Anti-tyrosinase Activity
The effect of inhibitor on mushroom tyrosinase was measured using L-tyrosine (1 mM) as the substrate. Hydroquinone (1 mM) was chosen as tyrosinase inhibitor. Both substrate and inhibitor were prepared in 0.1 M phosphate buffer pH 6.5. Inhibition of tyrosinase activity was tested in a reaction mixture (4 mL) containing 1.960 mL phosphate buffer, 2 mL L-tyrosine (1 mM), 20 μL mushroom tyrosinase and 20 μL hydroquinone (1 mM). The reaction was initiated by addition of enzyme to the solution of substrate and inhibitor. Then, the reaction cell and all solutions were both thermostated at 25 °C. Inhibition effect was determined by the diminution of the maximum quantity of dopachrome formed and the absorbance was measured spectrophotometrically at 475 nm. The inhibition percentage of tyrosinase activity was calculated as: Inhibition (%) = (A-B)/A × 100. Where A represents the optical density of the tyrosinase enzyme and B represents the optical density of the tested samples during 30 min. The assay was carried out in triplicate and Kojic acid was used as positive control agent. Sample concentration providing 50% inhibition (IC 50 ) was obtained plotting the inhibition percentage against sample concentrations [27].

Anti-butyrylcholinesterase Activity
Human plasma (pool plasma from samples designated for biochemical analysis) was used as a source of BChE. 100 μL of each sample were added to 100 μL of plasma and the mixture was incubated at 37 °C for 15 min. After incubation, the enzyme activity was measured by Konelab 30® UV apparatus at 405 nm. The control (plasma and distilled water) was treated in the same conditions. The assay was achieved in duplicate. Galanthamine was chosen as control positive. The anti-butyrylcholinesterase activity was calculated using the following formula: % Inhibition = [(Activity of control-Activity of sample)/Activity of control] × 100. The sample concentration providing 50% inhibition (IC 50 ) was determined by plotting inhibition percentages against concentrations of the sample. To evaluate temperature and incubation times effects, assays were performed by using the same procedure for the same sample, at 25 °C (fixed incubation times) and for different times at 37 °C [28].

Molecular Docking Procedure
The three-dimensional structures of PDB (PDB: 2Y9W) and PDB (PDB: 4TPK) were obtained from the RSCB protein data bank [29,30]. Before conducting the docking procedure, the original ligands and water molecules were removed. The polar hydrogens were then added to the enzyme structure. The optimization of all the geometries of scaffolds was performed with ACD (3D viewer) software (http://www.filef acts.com/acd3d -viewe r-freew are-info). Molecular docking of the studied chemical compounds 5a, 5c, 5d, 5e, and 5f at the tropolone-binding site was performed using autodock Vina software [31]. The analysis of intermolecular interactions has been performed using Pymol Version 0.99rc6.

Chemistry
We used DMF-DMA as a key reagent because of its high reactivity [25,26]. 4-Hydroxy-3-(quinolin-2-yl)-2Hchromen-2-one derivatives 5a-f were synthesized through a four-step reaction (Scheme 1). The 3-acetyl-4-hydroxycoumarin 2 obtained by acetylation of 4-hydroxycoumarin 1 was condensed with a series of primary aromatic amines, in ethanol, for 4 h to afford compounds 3. Compounds 4a-f, prepared by treating 3 with DMF-DMA in toluene for 10 min, were heated in acetic acid for 1 h to produce the target compounds 5a-f (Table 1). Mechanically, the formation of compound 5 (Scheme 2) starts by the protonation of dimethyl nitrogen in intermediates 4, the free doublet of nitrogen N 1′ being engaged in an intramolecular hydrogen bond with the OH group of the coumarin moiety. Cyclization of this intermediate was made possible by an intramolecular rearrangement similar to that of Diels-Alder reactions, thus producing the 5a-f derivatives after aromatization and departure of an NHMe2 molecule. The structures of substituted 4-hydroxy-3-(quinolin-2-yl)-2H-chromen-2-one derivatives 5a-f, were assigned on the basis of on their 1 H and 13 C NMR spectral data. The DCI-HRMS mass spectra of all the compounds were consistent with the proposed structures.

Biological
Compounds 4 and 5 were evaluated for their anti-tyrosinase and anti-butyrylcholinesterase activities.

Anti-tyrosinase Inhibitory
The anti-tyrosinase activity of compounds 4a-f and 5a-f was carried. The results clearly indicated that compounds 5a-f were more active than their precursors 4a-f, demonstrating the net contribution importance of the formed quinoline system, which appeared to be involved in the inhibition of tyrosinase (Table 2). Compounds 5a, 5c, 5e, and 5f were found to display remarkable tyrosinase inhibiting abilities (IC 50 = 17.5 ± 1.0, 18.3 ± 0.5, 17.9 ± 0.7 and 15.1 ± 0.8 μM, respectively) compared to the positive control kojic acid (IC 50 = 12.1 ± 0.2 μM). The compound 5f with a chlorine atom in C 7′ position showed the highest activity. The unsubstituted quinoline 5a displayed an important activity. This finding shows the importance of the chlorine atom in 5f which is certainly at the origin of the improvement of this activity. On the other hand, by comparing the activity of analogues 5b (R = Cl, R' = H) (IC 50 = 28.7 ± 1.1 μM) and 5f (R = H, R' = Cl), we can clearly see the influence of this position on the activity. Indeed, the chlorine in the meta-position was found to be more effective in terms of anti-tyrosinase activity. The inductive and mesomeric electronic effects exerted by the chlorine atom in each position could be at the origin of this difference in activity. The compound 5c with a methyl group at C 6′ , and the unsubstituted compound 5a, exhibited a comparable anti-tyrosinase effect, this suggests that the methyl group cannot be considered as a good candidate substituent which can improve this activity. Compound 5d with a methoxy at C 6′ was found to be less active (IC 50 = 24.9 ± 1.1 μM) than its analog 5e with an ethoxy group in the same position. This result shows clearly the Scheme 1 Synthesis of4hydroxy-3-(quinolin-2-yl)-2Hchromen-2-ones 5a-f. Reaction conditions: (i) Acetic anhydride, pyridine, 150 °C, reflux 2 h; (ii) Aromatic amines, EtOH, 79 °C, reflux 4 h; (iii) DMF-DMA (2 eq), Toluene, 110 °C, reflux 10 min; (iv) Acetic acid, 117 °C, reflux 1 h influence of the nature of the alkoxy group attached at C 6′ position on this activity.
The results described above clearly show the contribution of the quinoline fragment introduced to have this activity compared with that of the precursors 4a-f. This finding agrees well with the literature data showing the significant activity of quinoline derivatives, such as chloroquine [16] with a chlorine atom at the same position as the more active 5f derivative of the series 5.

Anti-butyrylcholinesterase Activity
Compounds 5a-f and their precursors 4a-f were assessed using an anti-butyrylcholinesterase test and the IC 50 values are indicated in Table 3. The compounds 4a-f were found to be less active than 5a-f ones. These results revealed the importance of cyclization, leading to the formation of quinoline, which appeared to be involved in the inhibition of BChE. The compound 5d with a methoxy group exhibited the highest anti-BChE effect with an IC 50 value of 40.0 ± 0.4 μM, followed by the derivative 5f with a chlorine atom at C 7′ (IC 50 = 51.0 ± 0.5 μM). The activity of the later compared to that of its analogue 5b with a chlorine atom at C 6′ (IC 50 = 89.0 ± 0.8 μM) allows to notice the influence of the position of the chlorine atom on this activity. The activity of these two chlorinated derivatives 5f and 5b compared to that of the unsubstituted compound 5a (IC 50 = 112.0 ± 2.0 μM), shows the importance of the chlorine atom whatever its position in improving this activity. The inductive and mesomeric electronic effects exerted by the chlorine atom in each position could explain this difference in activity. On the other hand, the compound 5d with a methoxy group at C 6′ showed twice the activity of its analogue 5e (IC 50 = 79.0 ± 0.5 μM) with an ethoxy group at the same position (IC 50 = 79.0 ± 0.5 μM). This showed that we may not need to extend the alkyl group of the alkoxy moiety to obtain better activity. Testing other compounds with longer alkoxy groups is necessary to provide greater support for this conclusion. The relatively weak activity of compound 5c with a methyl group at C 6′ (IC 50 = 99.0 ± 1.0 μM) when compared to the rest of the substituted derivatives leads to the conclusion that this methyl group is not much involved in the possible interactions between the ligand and the amino acids of the enzyme.

Molecular Docking Analysis for Anti-tyrosinase Activity (PDB: 2Y9W)
Tyrosinase (PDB code: 2Y9W) is a tetrameric protein composed of four chains (A, B, C, and D) with the sequence length of 391. This binuclear copper-containing enzyme catalyzes the conversion of monophenol (tyrosine) and o-diphenol (L-DOPA) to the corresponding o-quinone derivative [30]. Molecular modeling studies were carried out by using Autodock Vina software [31] to understand the interactions of synthesized compounds 5a-f within the hydrophobic binding pocket of tropolone (PDB: 2Y9W), and to investigate the binding modes and binding energies ( Table 4) that lead to the observed SARs and differences in IC 50 .
The analyses of binding affinities and molecular interactions for the 4-hydroxy-3-(quinolin-2-yl)-2H-chromen-2-one derivatives found 5a, 5c, 5e, and 5f were the most active. As Table 4 indicates, the values of binding energy of these derivatives are higher than that of the control (kojic acid).
The SARs of the anti-tyrosinase agents in Fig. 1 support that compound 5a exercises through its coumarinic fragment a pi sigma interaction (dark purple color) with VAL-A-248, a conventional hydrogen bond interaction (green color) with HIS-A-244 and by its quinoline fragment a pi-pi stacking (dark pink color) with PHE-A-264 and pi alkyl interaction (light pink color) with VAL-A-283. Derivative 5c forms some hydrophobic interactions with VAL-A-248 and VAL-A-283 (pi sigma), HIS-A-259 (pi-pi shaping (dark pink color)), HIS-A-263 (pi-pi stacking), SER-A-282 (amide pi stacking (dark pink color)), ALA-A-286 (pi alkyl interaction), VAL-A-248 (alkyl interactions (light pink color)) and with HIS-A-85 (carbon hydrogen bond (grey color)) ( Fig. 1). Further, compound 5e is involved in pi alkyl interactions  with VAL-A-248 and VAL-A-283. Besides, it displayed a pi-pi shaping with HIS-A-244, a pi sigma interaction with HIS-A-263 and carbon hydrogen bond with MET-A-280 (Fig. 1). The most effective anti-tyrosinase agent 5f having the lowest binding affinity (Table 4) established interactions with residues HIS-A-244, VAL-A-248, HIS-A-263 and PHE-A-264 and VAL-A-283. In details, 5f was strongly bound by the hydroxyl functional group with HIS-A-244 in conventional hydrogen bond interactions, pi sigma interaction with VAL-A-248, pi-pi stacking with PHE-A-264, alkyl and pi alkyl interactions with HIS-A-263, PHE-A-264 andVAL-A-283 (Fig. 2).

Molecular Docking Analysis for Anti-butyrylcholinesterase Activity (PDB: 4TPK)
To understand the anticholinesterase potential of derivative 5d, binding interactions between ligand and butyrylcholinesterase BChE (PDB: 4TPK (chain A)) were analyzed [32]. Molecular docking analysis was performed using Autodock Vina software [31]. Figure 3 showed that hydroxycoumarin is involved in conventional hydrogen bonding (green color) by its hydroxyl functional groups with SER-A-198 and pi-pi shaping interactions (dark pink color) with TRP-A-231 and PHE-A-329 besides to pi alkyl interaction (light pink color) with LEU-A-286. Further, methoxyquinoline ring forms amide pi stacking interaction (dark pink color) with GLY-A-116, pi-pi shaping interaction with TRP-A-82, pi donor hydrogen bond with THR-A-120 and carbon hydrogen bond (grey color) with GLN-A-67.

Conclusion
In summary, we develop here a simple and easy method to synthesize heterocyclic compounds in a short time and with good yields. This was achieved by including quinoline and 4-hydroxycoumarin moieties in their structure, using DMF-DMA as the main reagent. We examined the anti-tyrosinase and anti-butyrylcholinesterase activities of these prepared heterocycles and some of them exhibited interesting antityrosinase and butyrylcholinesterase activities. Molecular docking analyses lead to the conclusion that the quinoline moiety is essential for the build-up and improvement of anti-tyrosinase and anti-butyrylcholinesterase activities of conjugates 5a-f. In silico SAR studies were found in good agreement with biological evaluation showing that the nature of the substitute on the quinoline ring is essential to give significant binding interaction with amino acids of enzymes. The chlorine atom appears to be in favor with the activities studied. The diversification of its position and its number merit further study.